Table 1.

Cofactor content of different protein samples

Relevant host genotype	Protein	PLP	PMP	Pyruvate–PLP	% Adduct
WT	Alr	0.87 ± <0.01	ND	ND	N/A
ridA	Alr	0.70 ± <0.01	ND	0.11 ± <0.01	14%
WT	AlrR209S	0.46 ± <0.01	ND	ND	N/A
ridA	AlrR209S	0.29 ± <0.01	ND	0.54 ± <0.01	65%
WT	AlrR209E	0.05 ± <0.01	ND	0.19 ± <0.01	79%
ridA	AlrR209E	0.02 ± <0.01	ND	0.98 ± <0.01	98%
WT	AlrR209D	0.18 ± <0.01	ND	ND	N/A
ridA	AlrR209D	0.18 ± <0.01	ND	0.33 ± <0.01	65%
WT	AlrR209A	0.78 ± <0.01	ND	ND	N/A
ridA	AlrR209A	0.30 ± <0.01	ND	0.30 ± <0.01	50%
WT	IlvA	1.25 ± 0.04	ND	ND	N/A
ridA	IlvA	0.45 ± 0.02	ND	0.03 ± <0.01	6%
WT	IlvE	0.30 ± <0.01	0.33 ± <0.01	0.12 ± <0.01	27%
ridA	IlvE	0.07 ± <0.01	0.11 ± <0.01	0.72 ± <0.01	80%

The relevant proteins were expressed and purified from the indicated host strain. Cofactors were released from each sample with base treatment and separated on HPLC. The amount of each cofactor (nmol/nmol protein) is reported. The % adduct column reports the ratio of pyruvate–PLP to pyruvate–PLP + PLP. Proteins were expressed in cells grown in minimal glucose containing 1 mM glycine, 50 μM pyridoxine, and 15 μg/ml ampicillin with supplementation of 0.2% (wt/vol) L-arabinose to induce expression of the protein-encoding gene and 5 mM L-serine to induce 2AA production, as described in the text.

Abbreviations: N/A, not applicable; ND, not detected.